2018 Fiscal Year Research-status Report
Dynamic Character of the Ubiquitin System
| Project/Area Number |
18K14665
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| Research Institution | Kyoto University |
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Principal Investigator |
Walinda Erik 京都大学, 医学研究科, 助教 (80782391)
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| Project Period (FY) |
2018-04-01 – 2020-03-31
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| Keywords | Protein binding / Ubiquitin / Molecular recognition / Protein dynamics |
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| Outline of Annual Research Achievements |
1.Dynamics of free diubiquitin
Relaxation dispersion measurements of ubiquitin, linear, K48-linked, and K63-linked diubiquitin were successfully concluded. Among these molecules, only K48-linked diubiquitin showed significant relaxation dispersion (Rex > 1.5 s-1). An exception is very low temperature (280 K) where even monomeric ubiquitin shows a few residues with relaxation dispersion (N25, V70). We also conducted statistical analyses of ubiquitin-binding proteins registered in the protein database looking at the interface area between binding protein and proximal/distal ubiquitin subunit. As a goal for this fiscal year, the dynamics data from NMR will be combined with the statistical analyses in a publication in the near future.
2.Recognition kinetics of diubiquitin
At the start of the project we already had access to domain-specifically labeled samples of linear diubiquitin. However, we had to obtain NMR resonance assignments for a ubiquitin-binding protein. In the fiscal year 2018, we obtained resonance assignments for the NZF domain of HOIL-1L in both the free and the bound form (Biomol. NMR Assign., 2018). On the basis of these assignments, we started kinetic measurements by NMR relaxation dispersion experiments. Kinetic parameters such as on- and off-rates could be obtained in a site-specific manner. Thermodynamic data by isothermal titration experiments were also obtained. In this final year of the project, we will continue to obtain kinetic data to synthesize a final model of the recognition process of linear diubiquitin by HOIL-1L NZF from a kinetic perspective.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The research is progressing rather smoothly. So far, we have presented the results at national scientific meetings; the results will be disclosed in two separate publications upon finishing the project. However, for K63-linked diubiquitin no relaxation dispersion could be observed; thus adjustment of the research plan was required (see below).
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| Strategy for Future Research Activity |
For the case of K48-linked diubiquitin, in this fiscal year, we will continue to gather diubiquitin dynamics data from NMR, molecular dynamics. As a goal for this fiscal year, the dynamics data from NMR will be combined with the statistical analyses in a publication.
As for linear diubiquitin, in this final year of the project, we will continue to obtain kinetic data to synthesize a final model of the recognition process of linear diubiquitin by HOIL-1L NZF from a kinetic perspective. This will be published as a separate study.
For K63-linked diubiquitin no R2 relaxation dispersion data could be obtained. Therefore, we will try to use other methods that probe faster dynamics such as R1rho relaxation dispersion and perform the experiments at lower temperature.
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| Causes of Carryover |
Stable isotopes were less expensive than expected and by using the auto-induction technique, we could improve the efficiency of our protein yield / 1 l of medium.
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